Not applicable.
This invention provides a Togavirus-amplified retrovirus vector and a novel method for packaging a retroviral cassette that contains a heterologous nucleic acid, which is self-amplified in a packaging cell cytoplasm.
Retrovirus vectors are currently the most advanced system available for mammalian gene therapy. These vectors are preferred for gene therapy because of their unique life cycle, which is divided into two phases: RNA and DNA. Wild-type retroviruses infect cells with a single-stranded RNA genome. This viral RNA genome is reverse-transcribed into a double-stranded DNA provirus, which integrates into the host genome. The viral RNA genome is then transcribed in the nucleus from the provirus. For the most part, the provirus functions as a cellular gene after integration into the genome. This characteristic is desirable for gene therapy as it provides a relatively stable copy of the gene of interest in the host genome. This characteristic, however, also creates a drawback for the production of recombinant vectors used in gene therapy: The nuclear-based transcription of the RNA genome limits the sequences that can be included in a recombinant retrovirus vector, due to nuclear RNA processing of the viral genome prior to packaging. Thus, the nuclear-based RNA synthesis of the retroviral genome results in the removal of intron sequences and other flanking and regulatory sequences from the retroviral RNA.
Typically, recombinant retrovirus vectors are constructed as a DNA provirus having a heterologous expression cassette. These vectors, in the form of recombinant DNA plasmids, are transduced into packaging cells. The DNA vector is transcribed in the nucleus, and the resulting RNA genome is processed in the nucleus before moving to the cytoplasm for packaging. Thus, heterologous genes included in the vector are processed before they are packaged into virus particles for gene therapy. The heterologous genes in retrovirus vectors, therefore, do not usually include complex, more gene-like features such as introns, polyadenylation signals, and flanking sequences because of this drawback in the vector system. Furthermore, the production of full-length retrovirus RNA can be severely impaired by transcriptional polyadenylation signals and transcriptional stop signals in 3xe2x80x2 flanking regions. Finally, often cDNAs are not expressed well in the nucleus due to the absence of processing signals (Chuah et al., Hum. Gene Ther. 6: 1363-1377 (1995)).
Other strategies have been used to construct retrovirus vectors that contain complex heterologous genes, but these systems have been unsuccessful. Attempts to integrate gene-like constructs in reverse orientation have been difficult mostly due to instability of these constructs and low vector titers (Jonsson et al., Hum. Gene Ther. 6: 611-623 (1995); Cone et al., Science 236: 954-957 (1987); Cosset et al., J. Virol. 69: 7430-7436 (1995)). These intron and flanking sequences, however, are often essential for efficient, stable in vivo gene expression (see, e.g., Brinster et al., Proc. Natl. Acad. Sci. U.S.A. 85: 836-840 (1988); Palmiter et al., Proc. Natl. Acad. Sci. U.S.A. 87: 6024-6028 (1991)). This lack of complexity thus limits the use of retrovirus vectors for the expression of heterologous genes in gene therapy.
Alphaviruses are a genus of the Togavirus family that have a positive strand, single-stranded RNA genome. Unlike retroviruses, Alphaviruses carry out their intracellular activities, including replication, in the cytoplasm. Certain members of the Alphavirus family are well-characterized, in particular Semliki Forest virus (SFV) and Sindbis virus (Liljestrom and Garoff, Biotechnology 9: 1356-1361 (1991); Rice et al., J. Virol. 61: 3809-3819 (1987)). Semliki Forest virus (SFV)-based expression systems have been used to express heterologous genes. In the SFV system, viral replicase proteins are translated from the viral RNA genome. The replicase proteins recognize specific viral replicase promoters and amplify SFV RNA. Eukaryotic RNA polymerases and nuclear RNA processing machinery are not involved in the synthesis of SFV RNA. However, Alphaviruses do not provide an integrated copy of the heterologous gene of interest in the host genome. Therefore, there is a need for a vector that provides stable and efficient expression of heterologous genes.
The present invention provides Togavirus-amplified retrovirus vectors and a novel method of packaging heterologous nucleic acid sequences in a retroviral cassette using a Togavirus system. These heterologous nucleic acids can include, for example, full length genes, cDNAs, introns, flanking regions, polyadenylation signals, or other sequences that are normally incompatible with expression of the retrovirus RNA in the cell nucleus. This system liberates vector design from constraints imposed by nuclear RNA transcription. To achieve this result, nuclear transcription and processing of retrovirus RNA in the packaging cell must be avoided. By using Togavirus mediated RNA self-amplification in the cytoplasm, the present invention provides vectors and a method of bypassing the DNA provirus stage during packaging. This invention therefore permits the production of retrovirus vectors that contain complex heterologous genes or cDNAs. Thus, the vectors and method of the invention are useful for gene therapy. An additional advantage of the Togavirus-amplified retrovirus vector production system is that it is able to produce high titers of retrovirus particles, due to its self-amplification capabilities.
The method of the invention includes first, selecting a packaging cell that produces retroviral components, including reverse transcriptase, integrase, gag proteins, and envelope proteins. Second, the cell is transduced with RNA sequences. These RNA sequences encode: the replicase gene cluster of a Togavirus, which is operably linked to a ribosomal binding site recognized by ribosomes of the packaging cell; and a Togavirus replicase target sequence, which contains a replicase promoter operably linked to a replicase amplification region. The replicase amplification region consists of a subgenomic promoter and the retroviral packaging cassette. The cassette comprises R and U5 regions at the 5xe2x80x2 end, reverse transcriptase recognition sites, a retroviral packaging sequence, a eukaryotic expression cassette that contains a promoter operably linked to a heterologous nucleic acid, and U3 and R regions at the 3xe2x80x2 end of the cassette. The R, U5, and U3 regions, the primer binding site and the polypurine tract, and the packaging sequence are compatible with the retroviral components of the packaging cell. Finally, the packaging cell is cultured under conditions that permit the replicase gene cluster to be translated; that permit the replicase to amplify the expression cassette; and that permit the retroviral components to package the expression cassette into retrovirus vector particles.
In one embodiment of the invention, the retrovirus vector includes the replicase gene cluster and the replicase amplification region on the same ribonucleic acid.
In one embodiment, the cell is transduced with an SFV particle comprising the RNA sequences. In another embodiment, the cell is transfected with the RNA sequences.
In another embodiment of the invention, the retrovirus vector is a murine retrovirus vector.
In one embodiment of the invention, the Togavirus is an Alphavirus. In another embodiment, the Togavirus is a Semliki Forest virus.
In another embodiment of the invention, the packaging cell line is selected from the group consisting of PA317, GP+E86, and PHOENIX.
In one embodiment of the invention, the heterologous nucleic acid is a ribozyme or an antisense sequence. In another embodiment, the heterologous nucleic acid carries untranslated genomic regions. In yet another embodiment of the invention, the heterologous nucleic acid is a cDNA. In another embodiment, the heterologous nucleic acid encodes human clotting factor 9.
In one embodiment of the invention, the method further includes separating the retrovirus vector from the packaging cells. In another embodiment of the invention, the method includes the step of infecting a eukaryotic cell with the retrovirus vector.
The invention also provides a nucleic acid sequence with the elements of the retrovirus vector of the method, as described above.
In one embodiment of the invention, the nucleic acid sequence is an RNA.
In another embodiment of the invention, the nucleic acid sequence contains a replicase gene cluster from an Alphavirus, and in yet another embodiment the replicase gene cluster is from a Semliki Forest virus.
In one embodiment of the invention, the nucleic acid sequence includes R, U5, and U3 regions derived from a murine retrovirus.
In another embodiment of the invention, the nucleic acid sequence includes a heterologous nucleic acid the comprises genomic untranslated regions.
In yet another embodiment of the invention, the nucleic acid sequence includes a heterologous nucleic acid that is a cDNA, a ribozyme, or an antisense sequence. In another embodiment, the heterologous nucleic acid sequence encodes human clotting factor 9.